Medicament Containing Taraxacum Plant Root Extract for Treatment or Prevention of Cancer, and Method for Preparing Same

ABSTRACT

The present invention relates to an improved method for preparing a medicament comprising a Taraxacum plant root extract for the treatment or prevention of a cancer. In one aspect, the method comprises freezing Taraxacum plant root to obtain a frozen root stock, said freezing step being selected to effect at least partial disruption of one or more root cells; dry grinding the frozen root stock to obtain a ground root powder, wherein during said dry grinding step the frozen root stock is maintained at a grinding temperature below about 40° C.; steeping the ground root powder with a solvent to obtain a suspension having a liquid extract portion and a solid particle portion; and separating the liquid extract portion from the solid particle portion to provide a separated liquid extract for use in the medicament.

RELATED APPLICATIONS

This application is a continuation application of prior U.S. applicationSer. No. 14/377,418 filed Feb. 8, 2013 (incorporated herein byreference), which was submitted with a 371(c) date of Aug. 7, 2014pursuant to 35 USC § 371 as the U.S. national phase applicationcorresponding to International Patent Application No. PCT/CA2013/000114filed Feb. 8, 2013, which claims the benefit of 35 USC § 119(e) to U.S.Provisional Application Ser. No. 61/597,453 filed Feb. 10, 2012(incorporated herein by reference).

SCOPE OF THE INVENTION

The present invention relates to an improved method of preparing amedicament which includes a root extract of plants belonging to thegenus Taraxacum, and which is for treatment, amelioration or preventionof cancers. More particularly, the present invention relates to thepreparation of a pharmaceutical composition which includes Taraxacumplant root extracts for use in the treatment and/or prevention ofcancers, and preferable colon cancers, pancreatic cancer, skin cancerssuch as melanoma, and blood cancers such as chronic lymphoid leukemia,chronic myeloid leukemia, chronic monocytic myeloid leukemia andHodgkin's lymphoma.

BACKGROUND OF THE INVENTION

Plants of the genus Taraxacum, also commonly known as dandelions; aremembers of the Asteraceae family. These plants are commonly found intemperate zones of the Northern Hemisphere, and species of dandelionsinclude T. officinale, T. erythrospermum, T. albidum, T. japonicum, T.laevigatum, T. erythrospermum and T. californicum.

Dandelions are tap-rooted biennial or perennial herbaceous plants withan average length of 15 to 30 cm. The leaves are large, light to darkgreen in color and cluster in a rosette at the base of the plant. Theflowering stalks are upstanding and carries a solitary, terminalinflorescence. The florescence ranges from 7 to 15 mm in diameter and iscomposed of 140 to 400 yellow, ligulate florets. The fruits are conicalachenes, brown and crowned by a white, hairy papus, which allows theseeds to be distributed by wind over long distances.

Taraxacum plant roots often contain a variety of compounds includingsesquiterpenes, carotenoids, coumarins, flavonoids, phenolic acids,polysaccharides, eudesmanolidcs, triterpenes, sterols, steroids andothers. Specific examples of such compounds include gennacranolide,eudesmanolide, guaianolide, taraxacin, phenylpropanoid glycosides,taraxacoside, lactupircin, lutein, violaxanthin, esculin, scopolctin,quercetin, luteolin, rutin, chrysoeriol, caffeic acid, vanillic acid,syringic acid, ferulic acid, chlorogenic acid, chicoric acid,p-hydroxyphenylacetic acids, p-hydroxylbenzoic acid, inulin, glucans,mannans, prunasin, 11β, 13-dihydrolactucin, ixerin D, ainsliosidetaraxinic acid, β-glucopyranosyl, taraxinic acid, glucosyl ester, 11,13-dihydrotaraxinic acid, l′-glucoside, lactucopicrin, lactucin,cichorin, tetrahydroridentin B, taraxacolide-O-β-glucopyranoside,prunasin, dihydroconiferin, syringin, dihydrosyringin, taraxasterol,Ψ-taraxasterol, homo-taraxasterol, stigmatsterol, cycloartcnol,umbelliferone, taraxalisin, α-amyrin, β-amyrin, arnidiol, faradiol,lupeol, taraxol, taraxaserol, 3β-hydroxylup-18-ene-21-one, β-sitosterol,campesterol, lettucenin A, choline, mucilage, pectin, and taraxerol.

Dandelion extracts have been used in the past as for exampleantioxidants, diuretics, analgesics, anti-coagulants and anticanceragents. The publication “Evaluation of aqueous extracts of Taraxacumofficinale on growth and invasion of breast and prostate cancer cells”International Journal of Oncology 32 (2008): 1085-1090 to Sigstedtreports on the anticancer activity of crude extracts prepared from theleaves (“DLE”), flowers (“DFE”) or roots (“DRE”) of the dandelionspecies Taraxacum officinale. The crude dandelion extracts in Sigstedtwere prepared by 1) soaking 75 g. of dried plant parts in water for 24hours at room temperature; 2) filtering the resulting mixture to removeparticulate matter; and 3) lyophilizing the mixture to obtain a powder.Sigstedt observes that DLE reduced the growth of MCF-7/AZ breast cancercells, and not that of LNCaP C4-2B prostate cancer cells; and that bothDFE and DRE failed to influence cancer cell proliferation.

The publication “Anti-carcinogenic Activity of Taraxacum Plant. I” Biol.Pharm. Bull. 22.6 (1999): 602-605 to Takasaki relates to dandelion rootextracts prepared from the species Taraxacum japonicum. Takasakidescribes extracting dried roots (600 g) of T. japonicum plant threetimes with 3 L of methanol for five hours each, and then evaporating themethanol solution to afford 109 g of a methanol extract. Takasakiadditionally describes the preparation of a water extract obtained fromextracting 60 g of T japonicum roots with 0.38 L of water for 1 hour,and then lyophilizing the resulting solution. Takasaki describes thatthe methanol and water extracts inhibited initiation and promotion oftwo-stage chemical carcinogenesis.

In the separate publication “Anti-carcinogenic Activity of TaraxacumPlant II” Biol. Pharm. Bull. 22.6 (1999): 606-610, Takasaki describesanother dandelion root preparation of T. japonicum obtained fromextracting dried roots (6.7 kg) with 40 L of n-hexane three times for 8hours each to produce a 120.5 g extract.

Dandelion plant parts have been utilized to prepare extracts in variousforms including capsules and tinctures. Dandelion roots in particularhave been harvested for preparing “dandelion coffee” obtained bysteeping dried ground plant root in boiling water. It has beenappreciated that such conventional forms of dandelion extracts areordinarily associated with lower anticancer activities, inducing as lowas 10% cell death when introduced to a cancerous or tumor tissue.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a medicament orpharmaceutical composition for the treatment, amelioration and/orprevention of cancers, and which includes a Taraxacum plant rootextract, preferably in combination with a pharmaceutically acceptablecarrier, a diluent, a binding agent, an adjuvant and/or other anticanceragents.

A further object of the present invention is to provide a Taraxacumplant root extract which is suitable and/or beneficial for use as amedicament or human consumption.

A yet further object of the present invention is to provide a method ofpreparing a medicament or pharmaceutical composition having a Taraxacumplant root extract which includes one or more compounds useful for thetreatment, melioration and/or prevention of a cancer.

It has been appreciated that a Taraxacum plant root extract may beuseful in the treatment and/or prevention of cancers, and which mayinclude without restriction pancreatic cancers, colon cancers, bloodcancers and skin cancers. Such skin cancers may be melanoma, and suchblood cancers may be leukemia, such as but not limited to Hodgkin'slymphoma, chronic lymphoid leukemia, chronic myeloid leukemia andchronic monocytic myeloid leukemia.

In one possible method, a medicament for the treatment or prevention ofa cancer may be prepared by: freezing Taraxacum plant root to obtain afrozen root stock, said freezing step being selected to effect at leastpartial disruption of one or more root cells; dry grinding the frozenroot stock to obtain a ground root powder, wherein during said drygrinding step the frozen root stock is maintained at a grindingtemperature below about 40° C.; steeping the ground root powder with asolvent to obtain a suspension having a liquid extract portion and asolid particle portion; and separating the liquid extract portion fromthe solid particle portion to provide a separated liquid extract for usein the medicament.

Although not intended to be bound by theory, it was experimentally shownthat anticancer compounds contained in a Taraxacum plant root mayundergo a reduction in their activities if subject to an elevatedtemperature in a dry environment, although such effect is lesspronounced or absent in wet environments. The applicant has recognizedthat during an extract preparation process, a Taraxacum plant root mayunfavorably be left exposed to dry heat, resulting in the reduction andpossibly elimination of anticancer activities. A Taraxacum plant rootand its anticancer activity may be most vulnerable to the deactivatingeffects of dry heat during the grinding step where the plant roots, rootcells and cellular contents could be heated on contact with a rotatingelement of a grinder, such as a grinder blade. A number of experimentswere performed to show that loss of activities could occur above 40° C.,and a complete loss of anticancer activities may result from exposure toa temperature above 70° C.

In a preferred embodiment, the grinding temperature is kept below about0° C., more preferably below about −25° C., and most preferably belowabout 40° C.

Furthermore, it has been appreciated that the anticancer activity ofmedicament having a Taraxacum plant root extract may be improved ifprepared with ground plant root obtained from dry grinding rather thanwet grinding. Dry grinding is believed to provide improved and/or morecontrollable disruption of root cells, and thus greater amounts ofintracellular anticancer contents or compounds available for subsequentextraction steps. Preferably, the frozen root stock is dry ground to anaverage particle size of less than about 100 μm, more preferably lessthan about 50 μm and most preferably between about 1 μm and about 30 μm.

In a preferred aspect, a Taraxacum root extract may be especially usefulfor inclusion in a medicament for the treatment, amelioration orprevention of cancers when prepared with dandelion roots obtained fromdormant Taraxacum plants harvested before, or more preferably withinabout 90 days, and most preferably about 30 days prior to plant bloomingor budding in the spring season, or before entering dormancy in thewinter season when bud growth ceases.

Although not intended to be bound by theory, it is believed thatTaraxacum plant roots undergo physiological changes in preparation ofblooming or dormancy. Specifically, based on the experimental resultsobtained from the extracts prepared from dandelion roots (“DRE”)obtained-at three different time points (Spring, Summer and beginning ofFall), the extracts prepared from the roots harvested in early springand beginning of the fall period were shown to be the most effective ininducing cell death in cancer cells. In particular, dandelion rootsharvested in the province of Ontario, Canada in March, September andOctober were shown to be highly effective in inducing apoptosis ofcancer cells. It is believed that the anticancer compounds in the rootextract are synthesized in preparation for dormancy (during the coldweather), and which may be involved in inducing cell death andeliminating the aged cells in the plant in preparation for winter.

In a preferred embodiment, the Taraxacum plant root is, prior to thefreezing step, dried to a relative humidity of about 5% to 10%.Preferably the plant root is diced into root pieces, which may have anaverage dimension between about 0.2 cm and 1.0 cm.

The Taraxacwn plant root is preferably obtained from a Taraxacum speciesincluding but not limited to T. officinale, T. erythrospermum, T.albidum, T. japonicum, T. laevigatum, T. erythrospermum and T.californicum. Most preferably, the plant root is harvested from T.officinale or T. laevigatum collected from an open grassy area.

Preferably in the freezing step the plant root is contacted or submergedin liquid nitrogen, or alternatively, subjected to a freezingtemperature below 0° C., or more preferably between about −210° C. andabout −30° C., for about 5 minute to 24 hours or until substantiallyfrozen.

The dry grinding step may be carried out with a grinder, including butnot limited to a mortar and pestle, a pulverizer, an impingement grinderand a micronized milling machine to effect substantial disruption ofroot cells. To reduce exposure to elevated temperatures in a dryenvironment above 40° C., the grinder is preferably cooled, with forexample liquid nitrogen, to prevent heating on contact with the frozenroot stock or the resulting ground root powder. Preferably, the grinderis cooled below about −25° C., and more preferably below about −50° C.

To better effect the release of therapeutically active compounds locatedinside the root cells, the grinding step is most preferably performed todisrupt or break open the cells and release their inner contents.

The ground root powder is steeped or soaked in a liquid or solvent,preferably in a polar solvent, such as water at a soaking temperaturebetween about 5° C. and about 100° C., or most preferably at about 25°C. Other suitable solvents include but not limited to pentane,cyclopentane, cyclohexane, benzene, toluene, 1,4-dioxane, chloroform,diethyl ether, dichloromethane, tetrahydrofuran, ethyl acetate, acetone,dimethylfomiamide, acetonitrile, dimethyl sulfoxide, propylenecarbonate, formic acid, n-butanol, isopropanol, n-propanol, ethanol,methanol and acetic acid. The ground root powder is preferably soaked inthe liquid between about 5 minutes to about 24 hours, or more preferablybetween about 10 minutes and about 30 minutes with or without stirring.Most preferably, the ground root powder is soaked in water at 10 gground root/50 mL water, and is boiled between 10 minutes and 30minutes.

The liquid extract portion of the suspension may be separated from thesolid particle portion preferably by filtration and/or centrifugation.Preferably, centrifugation, if performed, is carried out between 5000×gto 8000×g to remove any excess fibers. Filtration is preferablyperformed using suction filtration and a paper filter. The paper filterpreferably has the pore size of less than or equal to about 0.45 μm, andmost preferably less than or equal to about 0.22 μm. In a most preferredembodiment, the filtration step is performed stepwise using paperfilters of decreasing pore sizes (such as 0.45 μm filter, followed by0.22 μm filter). One or more filters or filter papers utilized for thefiltration step may be configured to remove a bacteria.

The separated liquid extract obtained from the suspension is preferablyfreeze dried to an extract powder. Preferably, the freeze drying step isperformed at a temperature between about −80° C. and −40° C.

The extract powder may be included in the medicament together with apharmaceutically acceptable carrier, a diluent, a binding agent, anadjuvant and/or additionally anticancer agents. Such anticancer agentsmay include metformin, hydroxyurea, cyclophosphamide and/or etoposide.

The medicament preferably include a dosage form which contains theextract powder in a range about 5 mg/kg weight/day to about 1000 mg/kgweight/day, and preferably about 10 mg/kg weight/day to about 70 mg/kgweight/day. Alternatively, 0.2 to 200 g, preferably about 0.5 g to about70 g, and most preferably about 1 to 4 g of the extract powder ispreferably included in medicament form as a daily dosage.

In yet another aspect, the present invention provides a method forpreparing a medicament comprising a Taraxacum plant root extract fortreatment or prevention of a cancer, the method comprising the steps of:(1). freezing Taraxacum plant root to obtain a frozen plant root stock,said freezing step selected to effect at least partial disruption of oneor more root cells, wherein said Taraxacum plant root comprises adormant Taraxacum plant root harvested either prior to plant budding orblooming, or after cessation of bud growth; (2) dry grinding said frozenplant root stock to obtain a ground plant root powder with an averageparticle size of less than about 100 μm, and preferably less than about50 μm, wherein during said dry grinding step the frozen root stock ismaintained at a grinding temperature below about 40° C.; (3) soaking theground plant root powder in a solvent comprising one or both of ethanoland water to produce a mixture having a liquid solution portion and asolid portion; (4) separating the liquid solution portion from the solidportion; and (5) freeze drying the liquid solution portion to obtain theTaraxacum plant root extract as a dried extract powder, and optionallymixing the dried extract portion with one or more of a pharmaceuticallyacceptable carrier, a diluent, a binding agent, an adjuvant and ananticancer agent.

The dormant plant root is harvested within 90 days, and preferably about30 days, prior to first seasonal plant blooming or budding. The plantroot may be from a plant belonging to a species of T. officinale, T.erythrospermum, T. albidunt, T. japonicum, T. laevigatum, T.erythrospermum and T. californicum.

The plant root is preferably dried to a relative humidity of less thanabout 10% before freezing. In the following freezing step, the plantroot is preferably contacted or submerged in liquid nitrogen to anaverage freezing temperature between about −210° C. and about −30° C.

In the dry grinding step, the frozen root stock is ground preferably toan average particle size of less than about 50 μm, and more preferablybetween about 1 μm to about 30 μm. The dry grinding step may be carriedout with a grinder, including but not limited to a mortar and pestle, apulverizer, an impingement grinder and a micronized milling machine toeffect substantial disruption of one or more root cells. To reduceexposure to elevated temperatures in a dry environment, the grinder ispreferably cooled, with for example liquid nitrogen, to a temperaturebelow about −25° C., and preferably below about −50° C.

The solvent for use in the soaking step may additionally include one ormore of pentane, cyclopentane, cyclohexane, benzene, toluene,1,4-dioxane, chloroform, diethyl ether, dichloromethane,tetrahydrofuran, ethyl acetate, acetone, dimethylformamide,acetonitrile, dimethyl sulfoxide, propylene carbonate, formic acid,n-butanol, isopropanol, n-propanol, methanol or acetic acid. The soakingstep is most preferably performed at a soaking temperature between about5° C. and about 100° C., preferably for a period of about 5 minutes toabout 24 hours, with or without stirring.

Various techniques may be utilized for separating the liquid solutionportion from the solid portion in the mixture. Such techniques mayinclude but not limited to centrifugation and filtration.Centrifugation, if performed, is preferably carried out between 5000×gto 8000×g. Filtration, if used, is most preferably performed at leasttwice using at least two filters of different pore sizes, such as about0.45 μm and about 0.22 μm. For improved safety for human consumption,one or more filters or filter papers utilized for the filtration stepmay be configured to remove a bacteria.

In a preferred embodiment, the grinding temperature is below about 0°C., more preferably below about −25° C., and most preferably below about−40° C.

The medicament preferably include a dosage form which contains theTaraxacum plant root extract in a range about 5 mg/kg weight/day toabout 1000 mg/kg weight/day, and preferably about 10 mg/kg weight/day toabout 70 mg/kg weight/day. Alternatively, about 0.5 g to about 70 g, andpreferably about 1 to 4 g of the Taraxacum plant root extract ispreferably included in the medicament in a daily dosage form.

In aspect (1), the present invention provides a method for preparing amedicament for the treatment or prevention of a cancer, the methodcomprising: freezing Taraxacum plant root to obtain a frozen root stock,said freezing step being selected to effect at least partial disruptionof one or more root cells; dry grinding the frozen root stock to obtaina ground root powder, wherein during said dry grinding step the frozenroot stock is maintained at a grinding temperature below about 40° C.;steeping the ground root powder with a solvent to obtain a suspensionhaving a liquid extract portion and a solid particle portion; andseparating the liquid extract portion from the solid particle portion toprovide a separated liquid extract for use in the medicament.

In aspect (2), the current invention provides a method according toaspect (1), wherein said cancer is a colon cancer, a pancreatic cancer,a blood cancer or a skin cancer.

In aspect (3), the present invention provides a method according toaspect (1) and/or (2), wherein said cancer comprises said blood canceror said skin cancer, and is selected from the group consisting ofchronic lymphoid leukemia, chronic myeloid leukemia, chronic monocyticmyeloid leukemia, Hodgkin's lymphonia, and melanoma.

In aspect (4), the present invention provides a method according to anyone or more of aspects (1) to (3) in any combination, wherein prior tosaid freezing step, the method further comprises drying said plant rootto a relative humidity between about 5% to about 10%.

In aspect. (5), the present invention provides a method according to anyone or more of aspects (1) to (4) in any combination, wherein saidTaraxacum plant root comprises a dormant Taraxacum plant root harvestedeither prior to plant blooming or budding, or after cessation of budgrowth.

In aspect (6), the present invention provides a method according to anyone or more of aspects (I) to (5) in any combination, wherein saiddormant Taraxacum plant root is harvested within about 90 days, andpreferably about 30 days, prior to said plant blooming or budding.

In aspect (7), the present invention provides a method according to anyone or more of aspects (1) to (6) in any combination, wherein saidTaraxacum plant root is from a plant belong to a species selected fromthe group consisting of T. officinale, T. erythrospermum, T. albidum T.japonicum, T. laevigatum, T. erythrospermum and T. californicum.

In aspect (8), the present invention provides a method according to anyone or more of aspects (1) to (7) in any combination, wherein saidfreezing step comprises contacting or submerging the plant root inliquid nitrogen, or freezing the plant root to an average freezingtemperature between about −210° C. and about −30° C.

In aspect (9), the present invention provides a method according to anyone or more of aspects (1) to (8) in any combination, wherein said drygrinding step comprises dry grinding the frozen root stock to an averageparticle size of less than about 100 μm, and preferably less than about50 μm.

In aspect (10), the present invention provides a method according to anyone or more of aspects (1) to (9) in any combination, wherein said drygrinding step comprises dry grinding the frozen root stock with agrinder selected from the group consisting of a pulverizer, animpingement grinder and a micronized milling machine, and wherein thegrinder or a component thereof is cooled below about −25° C., andpreferably below about −50° C., to prevent heating on contact with thefrozen root stock or the ground root powder.

In aspect (11), the present invention provides a method according to anyone or more of aspects (1) to (10) in any combination, wherein saidsolvent comprises one or more of water, pentane, cyclopentane,cyclohexane, benzene, toluene, 1,4-dioxane, chloroform, diethyl ether,dichloromethane, tetrahydrofuran, ethyl acetate, acetone,dimethylformamide, acetonitrile, dimethyl sulfoxide, propylenecarbonate, formic acid, n-butanol, isopropanol, n-propanol, ethanol,methanol and acetic acid.

In aspect (12), the present invention provides a method according to anyone or more of aspects (1) to (11) in any combination, wherein saidsteeping step comprises soaking the ground root powder in water at asoaking temperature between about 2° C. and about 150° C., andpreferably 5° C. and 100 ° C., preferably for a period of about 5minutes to about 24 hours, with or without stirring.

In aspect (13), the present invention provides a method according to anyone or more of aspects (I1) to (12) in any combination, wherein saidseparation step comprises at least one of filtration and centrifugation,and wherein the filtration is performed once or more than once using aplurality of filters of same or different pore sizes, and thecentrifugation is performed at 5000×g to 8000×g.

In aspect (14), the present invention provides a method according to anyone or more of aspects (1) to (13) in any combination, wherein saidseparation step comprises filtering the suspension at least twice with afirst filter having a first pore size of about 0.45 μm and a secondfilter having a second pore size of about 0.22 μm, and wherein thesecond filter is selected to remove a bacteria.

In aspect (15), the present invention provides a method according to anyone or more of aspects (1) to (14) in any combination, wherein prior tothe dry grinding step, the method further comprises dicing said plantroot to produce a plurality of root pieces.

In aspect (16), the present invention provides a method according to anyone or more of aspects (1) to (15) in any combination, wherein saidgrinding temperature is below about 0° C.

In aspect (17), the present invention provides a method according to anyone or more of aspects (1) to (16) in any combination, wherein saidgrinding temperature is below about −25° C., and preferably below about−40° C.

In aspect (18), the present invention provides a method according to anyone or more of aspects (1) to (17) in any combination, wherein saidmethod further comprises freeze drying the separated liquid extract toobtain an extract powder, and optionally mixing the extract powder withone or more of a pharmaceutically acceptable carrier, a diluent, abinding agent, an adjuvant and an anticancer agent.

In aspect (19), the present invention provides a method according to anyone or more of aspects (1) to (18) in any combination, wherein saidanticancer agent is one or more of metformin, hydroxyurea,cyclophosphamide and etoposide.

In aspect (20), the present invention provides a method according to anyone or more of aspects (1) to (19) in any combination, wherein saidmedicament comprises a dosage form having the extract powder in a rangeof about 5 mg/kg weight/day to about 1000 mg/kg weight/day, andpreferably about 10 mg/kg weight/day to about 70 mg/kg weight/day.

In aspect (21), the present invention provides a method according to anyone or more of aspects (1) to (20) in any combination, wherein saidmedicament comprises a daily dosage form having the extract powder in arange of about 0.5 g to about 70 g, and preferably about 1 to 4 g.

In aspect (22), the present invention provides a method for preparing amedicament comprising a Taraxacum plant root extract for treatment orprevention of a cancer, the method comprising the steps of: (1) freezingTaraxacum plant root to obtain a frozen plant root stock, said freezingstep selected to effect at least partial disruption of one or more rootcells, wherein said Taraxacum plant root comprises a dormant Taraxacumplant root harvested either prior to plant budding or blooming, or aftercessation of bud growth; (2) dry grinding said frozen plant root stockto obtain a ground plant root powder with an average particle size ofless than about 100 μm, and preferably less than about 50 μm, whereinduring said dry grinding step the frozen root stock is maintained at agrinding temperature below about 40° C.; (3) soaking the ground plantroot powder in a solvent comprising one or both of ethanol and water toproduce a mixture having a liquid solution portion and a solid portion;(4) separating the liquid solution portion from the solid portion; and(5) freeze drying the liquid solution portion to obtain the Taraxacumplant root extract as a dried extract powder, and optionally mixing thedried extract portion with one or more of a pharmaceutically acceptablecarrier, a diluent, a binding agent, an adjuvant and an anticanceragent.

In aspect (23), the present invention provides a method according toaspect (22), wherein said cancer is a colon cancer, a pancreatic cancer,a blood cancer or a skin cancer.

In aspect (24), the present invention provides a method according toaspect (22) and/or (23), wherein said cancer is chronic lymphoidleukemia, chronic myeloid leukemia, chronic monocytic myeloid leukemia,Hodgkin's lymphoma, or melanoma.

In aspect (25), the present invention provides a method according toanyone or more of aspects (22) to (24) in any combination, wherein saiddormant Taraxacum plant mot is harvested within about 90 days, andpreferably about 30 days, prior to first seasonal plant blooming orbudding.

In aspect (26), the present invention provides a method according to anyone or more of aspects (22) to (25) in any combination, wherein saidTaraxacum plant root is from a plant belonging to a species selectedfrom the group consisting of T. officinale, T. erythrospermunt, T.albidum, T. japonicum, T. laevigatum, T. erythrospermum and T.californicum.

In aspect (27), the present invention provides a method according to anyone or more of aspects (22) to (26) in any combination, wherein prior tosaid freezing step, the method further comprises drying said plant rootto a relative humidity less than about 10%.

In aspect (28), the present invention provides a method according to anyone or more of aspects (22) to (27) in any combination, wherein saidfreezing step comprises contacting or submerging the plant root inliquid nitrogen to an average freezing temperature between about −210°C. and about −30° C.

In aspect (29), the present invention provides a method according to anyone or more of aspects (22) to (28) in any combination, wherein said drygrinding step comprises dry grinding the frozen root stock with agrinder to the average particle size of less than about 50 μm, and toeffect substantial disruption of said one or more root cells, thegrinder being selected from the group consisting of a pulverizer, animpingement grinder and a micronized milling machine, and wherein saiddry grinding step further comprises cooling said grinder to atemperature below about −25° C.

In aspect (30), the present invention provides a method according to anyone or more of aspects (22) to (29) in any combination, wherein saidsolvent further comprises pentane, cyclopentane, cyclohexane, benzene,toluene, 1,4-dioxane, chloroform, diethyl ether, dichloromethane,tetrahydrofuran, ethyl acetate, acetone, dimethylformamide,acetonitrile, dimethyl sulfoxide, propylene carbonate, formic acid,n-butanol, isopropanol, n-propanol, methanol or acetic acid.

In aspect (31), the present invention provides a method according to anyone or more of aspects (22) to (30) in any combination, wherein saidsoaking step comprises soaking the ground plant root powder in thesolvent at a soaking temperature between about 5° C. and about 100° C.,preferably for a period of about 5 minutes to about 24 hours, with orwithout stirring.

In aspect (32), the present invention provides a method according to anyone or more of aspects (22) to (31) in any combination, wherein saidseparation step comprises at least one of filtration and centrifugation,and wherein the filtration is performed once or more than once using aplurality of filters of same or different pore sizes, and thecentrifugation is performed at 5000×g to 8000×g.

In aspect (33), the present invention provides a method according, toany one or more of aspects (22) to (32) in any combination, wherein saidseparation step comprises filtering the mixture at least twice with afirst filter having a first pore size of about 0.45 μm and a secondfilter having a second pore size of about 0.22 μm, and wherein one orboth said filters are selected to remove a bacteria.

In aspect (34), the present invention provides a method according to anyone or more of aspects (22) to (33) in any combination, wherein theanticancer agent comprises one or more of metformin, hydroxyurea,cyclophosphamide and etoposide.

In aspect (35), the present invention provides a method according to anyone or more of aspects (22) to (34) in any combination, wherein prior tothe dry grinding step, the method further comprises dicing saidTaraxacum plant roots to produce a plurality of root pieces having anaverage dimension selected at between about 0.2 cm to 1.5 cm.

In aspect (36), the present invention provides a method according to anyone or more of aspects (22) to (35) in any combination, wherein saidgrinding temperature is below about 0° C.

In aspect (37), the present invention provides a method according to anyone or more of aspects (22) to (36) in any combination, wherein saidgrinding temperature is below about −25° C., and preferably below about−40° C.

In aspect (38), the present invention provides a method according to anyone or more of aspects (22) to (37) in any combination, wherein saidmedicament comprises a dosage form having the Taraxacum plant rootextract in a range of about 5 mg/kg weight/day to about 1000 mg/kgweight/day, and preferably about 10 mg/kg weight/day to about 70 mg/kgweight/day.

In aspect (39), the present invention provides a method according to anyone or more of aspects (22) to (38) in any combination, wherein saidmedicament comprises a daily dosage form having the Taraxacum plant rootextract in a range of about 0.5 g to about 70 g, and preferably about 1to 4 g.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the percentage of human acute T-cellleukemia (Jurkat) cells induced to apoptosis (y-axis) upon treatmentwith DRE of varying amounts (x-axis).

FIG. 2 is a bar graph showing the percent viability of Jurkat cells(y-axis) at varying concentrations of DRE (x-axis).

FIG. 3 is a line graph showing the percent viability of A375 humanmelanoma cells (y-axis) treated for 24, 48 or 72 hours with DRE ofvarying concentrations (x-axis).

FIG. 4 is a series of fluorescence microscope images of A375 humanmelanoma cells stained with Hoechst 33342 dye after 48-hour treatmentwith DRE at concentrations of 1 mg/mL, 2.5 mg/mL, 5 mg/mL, and 10 mg/mL,and including a control.

FIG. 5 is series of 400×-magnified images of MV-4-11 cells stained withHoechst or Annexin-V stain (top and bottom rows, respectively) after48-hour treatment with DRE at concentrations of 0.6 mg/mL, 1.0 mg/mL,2.5 mg/mL, and 5.0 mg/mL, and including a control.

FIG. 6 is series of 400×-magnified images of U-937 cells stained withHoechst or Annexin-V stain (top and bottom rows, respectively) after48-hour treatment with DRE at concentrations of 0.6 mg/mL, 1.0 mg/mL,2.5 mg/mL, and 5.0 mg/mL, and including a control.

FIG. 7 is series of 400×-magnified images of HL-60 cells stained withHoechst or Annexin-V stain (top and bottom rows, respectively) after48-hour treatment with DRE at concentrations of 0.6 mg/mL, 1.0 mg/mL,2.5 mg/mL, and 5.0 mg/mL, and including a control.

FIG. 8 is a bar graph showing the percentage of MV-4-11 cells induced toapoptosis (y-axis) after 48-hour treatment with DRE at concentrations of0.6 mg/mL, 1.0 mg/mL, 2.5 mg/mL, and 5.0 mg/mL, and including a control.

FIG. 9 is a bar graph showing the percentage of HL-60 cells induced toapoptosis (y-axis) after 48-hour treatment with DRE at concentrations of0.6 mg/mL, 1.0 mg/mL, 2.5 mg/mL, and 5.0 mg/mL, and including a control.

FIG. 10 is a bar graph showing the percentage of U-937 cells induced toapoptosis (y-axis) after 48-hour treatment with DRE at concentrations of0.6 mg/mL, 1.0 mg/mL, 2.5 mg/mL, and 5.0 mg/mL, and including a control.

FIG. 11 is a bar graph showing activation or activity of caspase-8 inMV-4-11 cells (y-axis) 5 minutes, 15 minutes, 30 minutes and 60 minutesafter treatment with 0.6 mg/mL of DRE, and including a control.

FIG. 12 is a bar graph showing activation or activity of caspase-3 inMV-4-11 cells (y-axis) 5 minutes, 15 minutes, 30 minutes and 60 minutesafter treatment with 0.6 mg/mL of DRE, and including a control.

FIG. 13 is series of images of PANC-1 cells stained with Hoechst dyetreated with DRE at concentrations of 2.5 mg/mL, 5 mg/mL and 7.5 mg/mLof DRE (rows) for 24 hours, 48 hours, 72 hours and 96 hours (columns),and including controls.

FIG. 14 is a bar graph showing average percent apoptosis of PANC-1 cells(y-axis) treated with DRE at concentrations of 0.5 mg/mL, I mg/mL, 2.5mg/mL, 5 mg/mL and 7.5 mg/mL for 24 hours, 48 hours, 72 hours and 96hours (x-axis), and including controls.

FIG. 15 is series of images of hematoxylin and eosin stained livertissue of balb/c mice at 40× or 63× magnification (top and bottom rows,respectively) after treatment with plain filter water or watercontaining 5.0 mg/mL of DRE for a month (left and right columns,respectively).

FIG. 16 is a line graph showing weights of balb/c mice (y-axis) treatedwith DRE at concentrations of 2.5 mg/mL or 5.0 mg/mL on different days(x-axis), and including controls.

FIG. 17 is a series of 400×-magnified microscope images of DnFADD cellsstained with Hoechst 33342 dye (upper row) or viewed under phasecontrast illumination (bottom row) after treatment with DRE atconcentrations of 0.4 mg/mL, 0.6 mg/mL, and 2.5 mg/mL for 96 hours, andincluding controls.

FIG. 18 is a bar graph showing average percent apoptosis of peripheralblood mononuclear cells (y-axis) treated with DRE at concentrations of0.4 mg/mL, 0.6 mg/mL, and 2.5 mg/mL (x-axis), and including controls.

FIG. 19 is a graph showing the concentrations of peripheral bloodmononuclear cells (y-axis) treated with DRE at concentrations of 0.4mg/mL and 0.6 mg/mL (x-axis), and including controls.

FIG. 20 is a graph showing the concentrations of DnFADD cells (y-axis)treated with DRE at concentrations of 0.6 mg/mL and 2.5 mg/mL (x-axis)as obtained using the trypan blue exclusion assay, and includingcontrols.

FIG. 21 is a bar graph showing average showing activation or activity ofcaspase-8 in DnFADD cells (y-axis) treated with DRE for 15 minutes, 30minutes, 60 minutes, 180 minutes and 1440 minutes, and includingcontrols.

FIG. 22 is a series of images of peripheral blood mononuclear cellsisolated from a newly-diagnosed leukemia patient, and stained withHoescht or Annexin-V stain (top and bottom rows, respectively) aftertreatment with DRE at concentrations of 1.0 mg/mL, 2.5 mg/mL and 5.0mg/mL, and including controls.

FIG. 23 is a bar graph showing the percentage of peripheral bloodmononuclear cells isolated from a newly-diagnosed leukemia patient, andinduced to apoptosis (y-axis) after 24-hour or 48-hour treatment withDRE at concentrations of 1.0 mg/mL, 2.5 mg/mL and 5.0 mg/mL, andincluding controls.

FIG. 24 is a bar graph showing viability percentage of HT-29 human coloncancer cells (y-axis) treated with DRE at concentrations of 0.5 mg/mL,1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 2.5 mg/mL, 3.0 mg/mL, 3.5 mg/mL, 4.0mg/mL, 4.5 mg/mL, 5.0 mg/mL, 5.5 mg/mL and 6.0 mg/mL for 24, 48, 72 and96 hours, and including controls.

FIG. 25 is series of images of hematoxylin and Eosin stained heart,kidney and liver tissues of balb/c mice after treatment with plaintfiltered water (upper rows) or with DRE for a month (bottom rows).

FIG. 26 is a line graph showing weights of balb/c mice (y-axis) treatedwith DRE on different days (x-axis), and including controls.

FIG. 27 is a bar graph showing the amount of protein (y-axis) detectedin urine samples collected from balb/c mice treated with DRE, andincluding controls.

FIG. 28 is a line graph showing weights of control CD-1 nu/nu mice(y-axis) on different days (x-axis).

FIG. 29 is line graph showing weights of CD-1 nu/nu mice (y-axis)treated with DRE at the concentration of 2.5 mg/mL on different days(x-axis).

FIG. 30 is a photograph of a CD-1 nu/nu mouse transplanted with HT-29cells, and treated with filtered plain filtered water for three weeks.

FIG. 31 is a photograph of CD-1 nu/nu mouse transplanted with HT-29cells, and treated with DRE at the concentration of 2.5 mg/mL for threeweeks.

FIG. 32 is a bar graph showing tumor volumes (y-axis) of CD-1 nu/nu micetreated with DRE at the concentration of 2.5 mg/mL on different days(x-axis), and including controls.

FIG. 33 is series of images of hematoxylin and eosin stained heart,kidney, liver and xenotransplanted tumor tissues of CD-1 nu/nu mice at10× or 63× magnification (top and bottom two rows, respectively) aftertreatment with plain filtered water for a month.

FIG. 34 is series of images of hematoxylin and eosin stained heart,kidney, liver and xenotransplanted tumor tissues of CD-1 nu/nu mice at10× or 63× magnification (top and bottom two rows, respectively) aftertreatment with DRE at the concentration of 2.5 mg/mL for a month.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A Taraxacum plant root extract for use in the treatment and/orprevention of a cancer, and in accordance with a preferred embodiment ofthe present invention, was prepared. To prepare the preferred Taraxacumplant root extract, dandelions of the species T. officinale werecollected in Ontario, Canada about 30 days prior to blooming in thespring season and right at the beginning of the fall season. Thecollected plants were washed in water and then cut at the base of thestem to harvest the roots. The harvested roots were then slicedlengthwise into pieces of approximately ¼″ in length.

The cut root pieces were immersed in liquid nitrogen for about 5 to 10minutes until thoroughly frozen. The frozen pieces were ground up in animpingement grinder to an average particle size of about ≤45 μm. Theground root was soaked in boiled distilled water for an hour to extractand solubilize the active compounds.

Following the extraction/solubilization step, the distilled watercontaining the active compounds was vacuum filtered using a paper filterwith a pore size of about 0.45 μm to remove other plant matters andexcess fibers. The resulting filtrate was then freeze dried at −80° C.to obtain a powdered root extract. The dried extract was reconstitutedin water to give a final concentration of 100 mg/ml stock sample. Thestock sample of root extract was further vacuum filtered with abacterial paper filter having a pore size of about 0.22 μm to sterilizeand prepare the extract for use. For administration, about 1 g of thepowdered root extract was resolubilized in about 10 mL of boiled waterand then filtered. The filtrate was then be orally administered to apatient diagnosed with cancer. Preferably, for oral administration theratio of powdered root extract to water, should be approximately in theamount of between about 0.1 g to 50 g per 100 mL.

Several fractions of the plant root extract of the present inventionwere isolated and tested for bioactivity testing. Based on the mechanismof apoptosis induced by DRE, multiple compounds may be responsible forthe activity either alone or together in combinations for one or moredifferent targets. Furthermore, DRE of the present invention was shownin in vitro studies, including those performed with leukemia, coloncancer, pancreatic cancer and melanoma, to selectively induce programmedcell death types I and II in human cancer cell lines, while retainingnon-cancerous cells unsusceptible to apoptosis and autophagy induction.In particular, the inventors have appreciated that DRE may induce celldeath by the rapid activation of the extrinsic cell death pathwaypossibly by targeting the death receptors, such as for example Fas orTNF family of death receptors, on cancer cells or activating the DeathInducing Signaling Complex, as evidenced by the rapid activation ofcaspase-8 and the subsequent activation of caspase-3, followingtreatment.

Furthermore, the compounds in DRE were shown to directly target themitochondria of cancer cells suggesting that there are components of DREthat directly interact with the mitochondria, causing itsdestabilization for the release of pro-apoptotic factors and thegeneration of reactive oxygen species. DRE is believed to containmultiple compounds that could possibly have multiple targets, and whichmay be present as water soluble salts, ligand analogs or otherinteracting/binding proteins.

The medicament of the present invention were tested with a number ofcell lines for its activity and/or safety. In addition, ex vivoexperiments were performed with cell lines isolated from ten differentcancer patients suffering from chronic lymphoid leukemia, chronicmyeloid leukemia or chronic monocytic myeloid leukemia. Blood samplescollected from the patients were treated with different doses of thedandelion root extract for 48 hours. When compared to blood cell linesisolated from healthy volunteers, the dandelion root extract was shownto induce apoptosis in cell lines of chronic lymphoid leukemia, chronicmyeloid leukemia and chronic monocytic myeloid leukemia

Further provided below is a Table which summarizes a number ofadditional tests performed on other cell lines and the experimentalresults obtained for each tested cell line:

Cell line designation Name EC₅₀ Result Jurkat E6-1 Acute T-cell leukemia120 μg/ml DRE is capable of inducing apoptosis at low concentrations inJurkat cells MV-4-11 Chronic Myelomonocytic 120 μg/ml DRE effectivelyinduces Leukemia apoptosis and pro-death autophagy in a dose and timedependent manner U-937 Acute Monocytic 120 μg/ml DRE effectively inducesLeukemia apoptosis in a dose and time dependent manner HL-60 Acute 120μg/ml DRE effectively induces Promyelomonocytic apoptosis in APL cellsLeukemia A375 Melanoma 500 μg/ml DRE has been very effective in inducingapoptosis in drug-resistant melanoma cells. This effect is enhanced bythe metabolism interfering drug, metformin Pane-1 Pancreatic cancer cellline 500 μg/ml DRE effectively induces apoptosis and pro-death autophagyin a dose and time dependent manner BxPC-3 Pancreatic cancer cell line500 μg/ml DRE effectively induces apoptosis and pro-death autophagy in adose and time dependent manner HT-29 Colorectal cancer cell line 200μg/ml DRE is effective in inducing apoptosis in aggressive colon cancercells PBMC Peripheral Blood 200 μg/ml Experiments have been doneMononuclear Cells (From using samples from 9 newly diagnosed patients.DRE effectively leukemia patients induced apoptosis in PBMCs obtainedfrom leukemia patients in a dose and time dependent manner

To further explicitly illustrate the effectiveness of the medicament ofthe present invention, detailed descriptions of exemplary experimentsare provided below:

i) Anticancer activity of dandelion root extract on human T-cellleukemia cells

The activity of DRE against a human acute T-cell leukemia cell line(Jurkat) was evaluated in parallel to its effect on non-cancerousperipheral blood mononuclear cells (PBMCs). As illustrated in FIG. 1,crude dandelion extract (100 μL) induced apoptosis in approximately 50%of the cells as determined by manual counting of Hoescht images.Further, as illustrated in FIG. 2 showing the effect of DRE on theviability or Jurkat cells at 0.4 and 0.6 mg/mL, as determined by WST-1cell proliferation assay, decreased cell viability was observed withincreasing concentrations of DRE. Our findings showed that DRE iscapable of selectively inducing apoptosis at low concentrationsspecifically in cancer cells with no toxicity to PBMCs. Furthermore, itwas shown that DRE treatment led to very early activation of caspase-8and subsequent activation of caspase-3.

ii) Anticancer Activity of Dandelion Root Extract on Aggressive HumanMelanoma Cells

The effect of DRE on human melanoma cell lines in vitro was studied. Formelanoma, a very aggressive, chemo-resistant form of skin-cancer, DREwas very effective in inducing apoptosis as illustrated in FIGS. 3 and4. To generate FIG. 3, A375 human melanoma cells were seeded on 96-wellplates (about 1000 cells/well) and treated at different concentrationsof DRE for 24, 48 and 72 hours. As shown in FIG. 4, typical apoptoticmorphology was observed in the A375 cells treated with DRE at varyingconcentrations up to 10 mg/mL for 48 hours. To generate the images ofFIG. 4, the cells were stained with Hoechst 33342 dye, and the imageswere taken on a fluorescence microscope. Brightly stained, condensedbodies indicate apoptotic nuclei.

DRE was shown to also target the mitochondria, generating reactiveoxygen species. Further, drug-resistant melanoma cells were made moresensitive to DRE treatment by the metabolism interfering drug,metformin.

ill) Anticancer Activity of Dandelion Root Extract on Aggressive HumanChronic Myelomonocytic Leukemia (CMML) Cells

The efficacy of DRE in more aggressive leukemia cell lines was assessedto determine its selectivity and efficacy in inducingapoptosis/autophagy in CMML cells. DRE was shown to effectively induceapoptosis and autophagy in a dose and time dependent manner as shown inFIGS. 5 to 10.

The rapid activation of caspase-8 and caspase-3 as shown in FIGS. 11 and12 through the activation of the extrinsic pathway of apoptosis, wasobserved in the CMML cells, comparable to levels found in Jurkat cells.To obtain the bar graphs of FIGS. 11 and 12, MV-4-11 cells werecollected following treatment with DRE at the indicated time points andDRE concentrations, washed and incubated with lysis buffer to obtaincell lysate. The cell lysate was incubated with caspase substratesspecific to each caspase and incubated for an hour. Fluorescencereadings were obtained using a spectrofluorometer.

As shown in FIGS. 17 and 20, jurkat cells expressing a dominant-negativeFADD (DnFADD) protein, a major component of the death-inducing signalingcomplex (DISC), were insensitive to apoptosis induced by DRE, furtherindicating involvement of the extrinsic pathway of cell death. FIG. 21shows the activation of caspase-8 in DnFADD cells using caspase-8specific substrate and fluorescence readings, after treatment with DREat various time points, and which was prepared.

It was furthermore shown that induction of apoptosis in chronicmyelomonocytic leukemia cells was hindered after pre-treatment with apan-caspase inhibitor, z-VAD-fmk.

Non-cancerous peripheral blood mononuclear cells (ncPBMCs), treated withdandelion root extract in parallel, were not susceptible to apoptosis,demonstrating the selectivity of dandelion root extract in cell culture.

Results from this study indicate that the dandelion root extract of thepresent invention is useful as a novel non-toxic alternative toconventional cancer therapy available today. In addition, it is alsouseful in combination with conventional therapies (with lowerconcentrations of toxic compounds) to enhance their effects in thetreatment of cancer.

iv) Anticancer activity of Dandelion Root Extract on aggressive humanPancreatic cancer cells:

The dandelion root extract of the current invention may induce apoptosisin a dose and time dependent manner in aggressive human pancreatic celllines (B×PC-3 and PANC-1). As shown in FIGS. 13 and 14, increases inbrightly stained, condensed nuclei indicative of apoptosis was observedwith increasing doses and duration following treatment with DRE. Manualquantification of Hoechst pictures of the PANC-1 cells showed increasesin average percent apoptosis in a dose and time dependent manner.

In parallel, similar experiments in non-cancerous Normal Human and FetalFibroblasts showed that DRE selectively targets human pancreatic cancercells, confirming results from previous studies. Early activation ofcaspase-8 and subsequent activation of caspase-3 indicated thatapoptosis induction by DRE is due to activation of the extrinsic pathwayof apoptosis.

DRE induced a pro-death form of autophagy in human pancreatic cancercells. This induction of autophagy corresponds with the destabilizationof the mitochondrial membrane potential, which was observed agertreatment with DRE. Through revival experiments, it was shown that thesignal to commit suicide was retained once the cells had been exposed toDRE.

v) Anticancer Activity of Dandelion Root Extract (DRE) on OtherAggressive Human Cancer Cells

The DRE of the present invention was shown to be effective in aggressivehuman colon cancer and neuroblastoma cells. As sown in FIG. 24, theviability of HT-29 human colon cells was affected by treatment with DREin a time and dose dependent manners. EC₅₀ was determined to be 3.0mg/mL at 96 hours. FIG. 24 was prepared from data gathered in a WST-1cell proliferation assay. In particular, HT-29 human colon cancer cellswere seeded on 96-well plates (about 5000 cells/well), and then treatedwith DRE at different concentrations for 24, 48, 72 and 96 hours.

vi) Evaluation of Toxicity of Andelion Root Extract in Mouse Models

The toxicity of the extract of the present invention in In vivo mousemodels in the absence of any cancers using male balb/c mice was studied.There was a control group on plain filtered water regimen, and two DREgroups; a low dose group, given 2.5mg/ml DRE (equivalent human dose of105 g/day for a 70 kg patient) in their drinking water and a high dosegroup, given 5.0mg/ml DRE in their drinking water. On an average, eachmouse consumed approximately 5 mL of drinking solution per day, whichtranslated to 500 mg/kg/day (low dose group, extracted from 5 g of driedroot with an extraction ratio of 1:10) and 1,000 mg/kg/day (high dosegroup, extracted from 10 g of dried root with the same extraction ratioof 1:10). Such doses were higher than what was necessary for apoptoticinduction in the in vitro studies. These mice were given DRE in theirwater every day and monitored over one month, with the weights beingmeasured every other day as shown in FIGS. 16 and 26 of two separateexperiments. Following 34 days, the mice were sacrificed according tothe Animal Care Committee guidelines of the University of Windsor andthe organs (liver, kidneys and heart) were removed for pathologicalanalysis. As further shown in FIGS. 15 and 25, no toxicity on these micewere seen on the basis of measured weight and pathology. There was nodifference between the control untreated mice and the DRE-fed mice interms of weight change and pathology of the organs obtained.

Further efficacy studies were performed with four mice in the DREtreated group that were given 500 mg/kg/day of DRE for a total of 35days. Their tissues from liver, kidneys and heart were analyzed for anytoxic indication. The tissues did not show any change, compared to thewater-fed control mice. For further toxic indications, urine was alsoobtained from each group of mice and analyzed for protein content, usinga Bradford protein estimation assay. As shown in FIG. 27, lower levelsof protein were found in the DRE-fed mice, compared those of the controlmice. These results indicate that DRE of the present invention isnon-toxic and well-tolerated in mice, as a supplement to their drinkingwater, over a long period of time.

Further toxicity tests performed in vivo with mouse models confirmedthat the extract of the present invention does not present anysignificant toxicity at daily doses as high as 3% body weight,1.0g/kg/day or 100 g/day.

Based on the toxicity tests, the effective dosage for human patients maypreferably be about 0.5 to 4.0 g/day/patient (with 70 kg weight), ormore preferably 2.0 g/day/patient (which is less than 2% ofwell-tolerated dose in mice). One human patient who was treated with theDRE of the present invention was tolerant and responsive to 23mg/kg/day.

vii) Anticancer Activity of Dandelion Root Extract in Patient-DerivedEx-Vivo Samples of Leukemia

The effect of DRE in patient-derived leukemia samples from newlydiagnosed patients were studied. The experiment was performed usingsamples from 9 patients. Blood samples were obtained from newlydiagnosed patients and peripheral blood mononuclear cells (PBMCs) wereisolated and treated with the DRE of the current invention. As shownFIGS. 22 and 23, the DRE of the present invention effectively inducedapoptosis in PBMCs obtained from leukemia patients in a dose and timedependent manner. FIG. 23 was obtained by manual quantification ofHoechst pictures from six different patients.

viii) Efficacy of Dandelion Root Extract Against Human Colon CancerXenotransplant in Immunocompromised Mice

To evaluate the efficacy of DRE of the present invention in in vivomodels of various cancers, xenotransplants of colon cancer models weremade using immunocompromised CD-1 nu/nu mice. In particular, HT-29 cellswere injected on either side of the mice underneath the skin, andallowed to form tumors for a week prior to commencing treatments. Themice were divided into two groups (four mice per group), one on plainfiltered water regimen and the other was given 2.5 mg/mL aqueous DRE(400 mg/kg/day extracted from 5 g of dried root with an extraction ratioof 1:10) in their drinking water for a month. The weight of each mousewas obtained every other day and following a month of treatment, themice were sacrificed and the organs were obtained for pathologicalanalysis.

As shown in FIGS. 28 and 29, no differences in weights between thecontrol, water-fed mice and the DRE fed mice, confirming lack oftoxicity. FIGS. 30 and 31, respectively, are photographs of the CD-1nu/nu mice after three weeks of treatment with plain filtered water orDRE. As shown in FIG. 32, water-fed mice had larger tumor volumescompared to the DRE-fed mice, indicating the efficacy of DRE againstcolon cancer in in vivo models.

As further shown in FIGS. 33 and 34, tissue histochemical state of heartkidney and liver do not show any difference between control andDRE-treated animals indicating no toxicity to these tissues. On theother hand, there is clear difference in the tumor histochemistry ofcontrol and treated animals where significant decrease in the number oftumor cell nuclei could be seen.

Similar studies were done using IICT116 cells instead of HT-29 cells,and showed similar efficacy and toxicity results.

The above results indicate that DIRE was able to halt the growth ofcolon tumors in the DRE treatment group, compared to the water-fedgroups. There was no toxicity observed in the DRE treated groups,confirming the toxicity evaluation results. These results suggest thepotential efficacy of DRE in in vivo models of colon cancer.

ix) Clinical Data

A 70 year old man with refractory M5 acute myeloid leukemia reported tohave achieved a sustained remission lasting over 18 months with DREalone. Although he obtained complete remission from his acute monocyticleukemia, he continued to have evidence of chronic mylelomonocyticleukemia (CMML). His peripheral monocyte count was seen to rise when hedecreased his frequency of DRE consumption and similarly was controlledwhen he increased the amount of DRE consumed. Temporary responses in twowomen with chronic myelomonocytic leukemia, using DRE alone was alsoobserved.

Transient responses in patients consuming this product were reported.One patient with refractory acute myeloid leukemia started DRE andhydroxyurea at the same time, with immediate and dramatic response tothis combination. The patient had multiple large skin nodules that wentinto remission within 24 hours. The patient maintained this response forone month, despite stopping the hydroxyurea after only 24 hours. Hetolerated the drug extremely well, with no reported toxicity.

Another patient took the DRE for refractory Hodgkin's lymphoma. Thepatient was a 40 year female who failed multiple chemotherapies andautologous stem cell transplant. The patient undertook concomitantchemotherapy in the form of cyclophosphamide and etoposide. She sufferedfrom cytopenias from this combination, but was suffering cytopenia fromthese medications before the DRE was added. She had a dramatic, buttemporary response on CT scan when the DRE was added. She progressedafter three months on the product, and developed pancreatitis followingthis progression.

Many patients having used DRE for various malignancies includingcolorectal cancer reported excellent tolerance, and self-reportedresponses. Another patient with Hodgkin's lymphoma reported an apparentremarkable response to the treatment with DRE.

Other anticancer ingredients or drugs, which do not impair the functionsof the root extract may be added to the medicament of the presentinvention. Such anticancer ingredients may include, but not limited to,an antifolate, a 5-fluoropyrimidine (including 5-fluorouracil), acytidine analogue such as β-L-1,3-dioxolanyl cytidine orβ-L-1,3-dioxolanyl 5-fluorocytidine, antimetabolites (including purineantimetabolites, cytarabine, fudarabine, floxuridine, 6-mercaptopurine,methotrexate, and 6-thioguanine), hydroxyurea, mitotic inhibitors(including CPT-11, Etoposide (VP-21), taxol, and vinca alkaloids such asvincristine and vinblastine), an alkylating agent (including but notlimited to busulfan, chlorambucil, cyclophosphamide, ifofamide,mechlorethamine, melphalan, and thiotepa), nonclassical akylatingagents, platinum containing compounds, bleomycin, an anti-tumorantibiotic, an anthracycline such as doxorubicin and dannomycin, ananthracenedione, topoisomerase II inhibitors, hormonal agents (includingbut not limited to corticosteriods (dexamethasone, prednisone, andmethylprednisone), androgens such as fluoxymesterone andmethyltestosterone), estrogens such as diethylstilbesterol,antiestrogens such as tamoxifen, LHRH analogues such as leuprolide,antiandrogens such as flutamdie, aminogluetethimide, megestrol acetate,and medroxyprogesterone, asparaginase, carmustine, lomustine,hexamethyl-melamine, dacarbazine, mitotane, streptozocin, cisplatin,carboplatin, levamasole, and leucovorin. Preferably, the anticanceragent is metformin, hydroxyurea, cyclophosphamide or etoposide. Thecompounds of the present invention can also be used in combination withenzyme therapy agents and immune system modulators such as aninterferon, interleukin, tumor necrosis factor, macrophagecolony-stimulating factor and colony stimulating factor. The rootextract may be administered to a patient by any appropriate route which,for example, may include oral, parenteral, intravenous, intradermal,transdermal, mucosal, subcutaneous, and topical.

Preferably, the root extract is administered orally. A number ofadministration/dosage experiments showed that the medicament of thepresent invention may produce greater anticancer activity if ingestedorally, and possibly exposed to the subject's digestive system. The rootextract may be orally administered in powder or liquid extract formwithout further modifications. Alternatively, the root extract may besolubilized in a liquid, most preferably in water, the liquid containingthe extract is orally administered. To prevent inadvertent introductionof a bacteria or bacterial infection, the extract of the presentinvention may be boiled into a tea and the tea containing the extractmay be orally administered. The root extract may alternatively beenclosed in capsules or compressed into tablets. Such capsules ortablets may be purified to remove impurities and/or bacteria, or furtherinclude an inert diluent, an edible carrier, binding agents, and/oradjuvant materials.

The tablets, capsules, and the like can contain any of the followingingredients, or compounds of similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose; a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. When the dosage unitform is a capsule, it can contain, in addition to the aforementionedmaterials, a liquid carrier such as fatty oil. In addition, dosage unitforms can contain various other materials which modify the physical formof the dosage unit, for example, coating of sugar, shellac, or otherenteric agents.

It is to be noted that dosage will vary with the conditions, age, bodyweight and severity of the cancer to be treated. It will be readilyapparent to a person skilled in the art that for each patient, specificdosage regimens could be adjusted over time according to individualneeds. The root extract may be administered once or may be divided intoa number of smaller doses to be administered at varying intervals oftime.

The medicament of the present invention is suitable for treatment and/orprevention of a cancer, including that of skin tissues, organs, bone,cartilage, blood and vessels. The root extract may be used to treatvariety of cancers including, but not limited to, cancer of the head,neck, eye, mouth, throat, esophagus, chest, bone, lung, colon, rectum,stomach, prostate, breast, ovaries, kidney, liver, pancreas and brain.The cancer encompasses primary and metastatic cancers.

The most preferred embodiments of the present invention are describedhereto. The most preferred embodiments are provided as mere exampleswhich arc in no way intended to limit the scope of the presentinvention. It will be readily apparent to a person skilled in the artthat variations and modifications may be made to the most preferredembodiments within the scope of the present invention.

We claim:
 1. A method for preparing a medicament for the treatment orprevention of a cancer, the method comprising: freezing Taraxacum plantroot to obtain a frozen plant root stock, said freezing step beingselected to effect at least partial disruption of one or more rootcells; dry grinding the frozen plant root stock to obtain a ground plantroot powder, wherein during said dry grinding step the frozen root stockis maintained at a grinding temperature below about 40° C.; soaking orsteeping the ground plant root powder with a solvent comprising one ormore of water, methanol, ethanol, n-propanol, isopropanol and n-butanolto obtain a mixture having a liquid extract portion and a solid particleportion; and separating the liquid extract portion from the solidparticle portion to provide a separated liquid extract for use in themedicament.
 2. The method of claim 1, wherein said cancer is a coloncancer, a pancreatic cancer, a blood cancer or a skin cancer.
 3. Themethod of claim 2, wherein said cancer comprises said blood cancer orsaid skin cancer, and is selected from the group consisting of chroniclymphoid leukemia, chronic myeloid leukemia, chronic monocytic myeloidleukemia, Hodgkin's lymphoma, and melanoma.
 4. The method of claim 1,wherein prior to said freezing step, the method further comprises dryingsaid plant root to a relative humidity between about 5% to about 10%. 5.The method of claim 1, wherein said Taraxacum plant root is a dormantTaraxacum plant root harvested prior to plant blooming or budding orafter cessation of bud growth.
 6. The method of claim 5, wherein saiddormant Taraxacum plant root is harvested within about 90 days prior toa blooming or budding season of the Taraxacum plant.
 7. The method ofclaim 1, wherein said Taraxacum plant root is from a plant belong to aspecies selected from the group consisting of T. officinale, T.erythrospermum, T. albidum, T. japonicum, T. laevigatum, T.erythrospermum and T. californicum.
 8. The method of claim 1, whereinsaid freezing step comprises contacting or submerging the plant root inliquid nitrogen, or freezing the plant root to an average freezingtemperature between about −210° C. and about −30° C.
 9. The method ofclaim 1, wherein said dry grinding step comprises dry grinding thefrozen plant root stock to an average particle size of less than about100 μm.
 10. The method of claim 1, wherein said dry grinding stepcomprises dry grinding the frozen plant root stock with a grinderselected from the group consisting of a pulverizer, an impingementgrinder and a micronized milling machine, and wherein the grinder or acomponent thereof is cooled below about −25° C. to prevent heating oncontact with the frozen plant root stock or the ground plant rootpowder.
 11. The method of claim 10, wherein the grinder or a componentthereof is cooled with liquid nitrogen.
 12. The method of claim I,wherein said solvent comprises one or more of water, pentane,cyclopentane, cyclohexane, benzene, toluene, 1,4-dioxane, chloroform,diethyl ether, dichloromethane, tetrahydrofuran, ethyl acetate, acetone,dimethylformamide, acetonitrile, dimethyl sulfoxide, propylenecarbonate, formic acid, n-butanol, isopropanol, n-propanol, ethanol,methanol, and acetic acid.
 13. The method of claim 1, wherein saidsolvent is water, ethanol or a mixture of water and ethanol.
 14. Themethod of claim 1, wherein said soaking or steeping step comprisessoaking the ground plant root powder in water at a soaking temperaturebetween about 5° C. and about 100° C. for a period of about 5 minutes toabout 24 hours with or without stirring.
 15. The method of claim 1,wherein said separation step comprises at least one of filtration andcentrifugation, and wherein the filtration is performed once or morethan once using a plurality of filters of same or different pore sizes,and the centrifugation is performed at 5000×g to 8000×g.
 16. The methodof claim 1, wherein said separation step comprises filtering the mixtureat least twice with a first filter having a first pore size of about0.45 μm and a second filter having a second pore size of about 0.22 μm,and wherein one or both said filters are selected to remove a bacteria.17. The method of claim 1, wherein prior to The dry grinding step, themethod further comprises dicing said plant root to produce a pluralityof root pieces having an average dimension selected between about 0.2 cmand about 1.5 cm.
 18. The method of claim 1, wherein said grindingtemperature is below about −25° C., or below about −40° C.
 19. Themethod of claim 1, wherein said method further comprises freeze dryingthe separated liquid extract to obtain an extract powder, and optionallymixing the extract powder with one or more of a pharmaceuticallyacceptable carrier, a diluent, a binding agent, an adjuvant and ananticancer agent.
 20. The method of claim 19, wherein said anticanceragent is one or more of metformin, hydroxyurea, cyclophosphamide andetoposide.
 21. The method of claim 19, wherein said medicament comprisesa dosage form having the extract powder in a range of about 5 mg/kgweight/day to about 1000 mg/kg weight/day.
 22. The method of claim 19,wherein said medicament comprises a daily dosage form having the extractpowder in a range of about 0.5 g to about 70 g.
 23. The method of claim6, wherein said dormant Taraxacum plant root is harvested about 30 daysprior to said blooming or budding season.